U.S. patent application number 09/923853 was filed with the patent office on 2002-05-02 for cell adhesion solution.
Invention is credited to Fox, Willaim Alan.
Application Number | 20020051849 09/923853 |
Document ID | / |
Family ID | 25449358 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020051849 |
Kind Code |
A1 |
Fox, Willaim Alan |
May 2, 2002 |
Cell adhesion solution
Abstract
A cell adhesion solution of a cross-linked polymer for
substrates includes an amino acid polymer, a buffered cross-linking
agent, and deionized water (DI H.sub.2O). In one example, the amino
acid polymer is selected from a neutral or basic amino acid, such
as poly 1-lysine or poly 1-arginine. In another example, the amino
acid polymer includes poly 1-1sine. In another example, the amino
acid polymer includes poly 1-1sine of molecular weight of 100,000
or higher. The cross-linking agent can be selected from an aldehyde
functional group, a buffered formaldehyde solution or a 10% neutral
buffered formalin solution.
Inventors: |
Fox, Willaim Alan;
(Burlington, NC) |
Correspondence
Address: |
GEORGE A LEONE, SR
2150 128TH AVENUE, NW
MINNEAPOLIS
MN
55448
US
|
Family ID: |
25449358 |
Appl. No.: |
09/923853 |
Filed: |
August 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60224469 |
Aug 10, 2000 |
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Current U.S.
Class: |
427/398.5 ;
427/443.2; 562/562 |
Current CPC
Class: |
C12N 5/0068 20130101;
C12N 2533/32 20130101 |
Class at
Publication: |
427/398.5 ;
427/443.2; 562/562 |
International
Class: |
B05D 003/00 |
Claims
What is claimed is:
1) A solution of a cross-linked polymer for substrates, said
solution comprising: a) an amino acid polymer; b) a buffered
cross-linking agent; and c) deionized water (DI H.sub.2O).
2) The solution of claim 1 wherein said amino acid polymer is
selected from the group consisting of a neutral amino acid, basic
amino acid, poly 1-lysine and poly 1-arginine.
3) The solution of claim 1 where said amino acid polymer comprises
poly 1-sine.
4) The solution of claim 1 where said amino acid polymer comprises
poly 1-1sine of molecular weight of 100,000 or higher.
5) The solution of claim 1 where said cross-linking agent comprises
an aldehyde functional group.
6) The solution of claim 1 where said cross-linking aldehyde agent
comprises a buffered formaldehyde solution.
7) The solution of claim 1 where said cross-linking aldehyde agent
comprises at least a 10% neutral buffered formalin solution.
8) A solution of a cross-linked polymer that creates strong and
stable cell adhesion properties to substrates when applied, said
solution comprising, by volume: a) up to 0.03% poly 1-lysine in DI
H.sub.2O; and b) up to 25% neutral buffered formalin.
9) The solution of claim 8 comprising up to 0.015% poly 1-lysine in
DI H.sub.2O and up to 12.5% neutral buffered formalin.
10) The solution of claim 8 comprising up to 0.0075% poly 1-lysine
in DI H.sub.2O and up to 6.25% neutral buffered formalin.
11) A slide preparation method for preparing a solution of a
cross-linked polymer for substrates, said method comprising the
steps of: a) mixing an amino acid polymer, a buffered cross-linking
agent; and deionized water (DI H.sub.2O); b) letting the mixture
stand for a predetermined time period forming a cell adhesion
solution; and c) transferring the cell adhesion solution to a
container for coating of substrates. d) dipping a substrate into
the cell adhesion solution; and e) drying the substrate by removing
the substrate from the cell adhesion solution.
12) The method of claim 11 wherein the drying step takes from 5
minutes to 1 hour.
13) The method of claim 11 wherein said amino acid polymer is
selected from the group consisting of a neutral amino acid, basic
amino acid, poly 1-lysine and poly 1-arginine The method of claim
11 wherein the amino acid polymer comprises poly 1-1sine.
14) The method of claim 11 wherein said amino acid polymer
comprises poly 1-1-sine of molecular weight of 100,000 or
higher.
15) The method of claim 11 wherein said cross-linking agent
comprises an aldehyde functional group.
16) The method of claim 11 wherein said cross-linking aldehyde
agent comprises a buffered formaldehyde solution.
17) The method of claim 11 wherein said cross-linking aldehyde
agent comprises at least a 10% neutral buffered formalin solution.
Description
RELATED APPLICATION
[0001] This application is related to co-pending provisional
application of William Alan Fox, Ser. No. 60/224,469, filed Aug,
10, 2000, entitled "CELL ADHESION SOLUTION," and, by this
reference, claims the benefit of the priority filing date of the
co-pending provisional application.
FIELD OF THE INVENTION
[0002] This invention relates to a liquid solution for coating
substrates that demonstrates long-term stability cell adhesion
properties and, more particularly, to a solution including a
cross-linked amino acid polymer for applying to cytological
specimen slides.
BACKGROUND OF THE INVENTION
[0003] Many diagnostic assays depend on the evaluation of
cytological and histological components. One requirement of these
evaluations is the adhesion of these components to a solid
substrate, such as a glass or plastic microscope slide. Once
immobilized by adhesion, these components may be processed further
to gain diagnostic information.
[0004] Several cytological/histological adhesion reagents have been
developed and used in the past, such as albumin, silane, gelatin,
and poly 1-lysine. Although all are effective adhesive reagents to
some degree, their adhesive properties do not remain stable once
coated, applied, and dried on a solid substrate. This results in
the need to prepare freshly coated slides for optimal routine
use.
SUMMARY OF THE INVENTION
[0005] The present invention provides a solution of a cross-linked
polymer for substrates. The solution includes an amino acid
polymer, a buffered cross-linking agent, and deionized water (DI
H.sub.2O).
[0006] In one embodiment, the amino acid polymer, as employed in
the invention, is selected from a neutral or basic amino acid, such
as but not limited to poly 1-lysine or poly 1-arginine.
[0007] In another embodiment, the amino acid polymer, as employed
in the invention, comprises poly 1-1sine.
[0008] In another embodiment, the amino acid polymer, as employed
in the invention, comprises poly 1-1sine of molecular weight of
100,000 or higher.
[0009] In another embodiment, the cross-linking agent, as employed
in the invention, comprises an aldehyde functional group.
[0010] In another embodiment, the cross-linking agent, as employed
in the invention, comprises a buffered formaldehyde solution.
[0011] In one aspect, the present invention provides a
cytological/histological adhesion reagent that retains its adhesive
properties in a stable fashion when applied to a solid substrate
such as a microscope slide.
[0012] In another aspect of the invention, it has been found that
poly 1-lysine is a suitable cytological adhesion reagent when
applied and dried on a glass slide, but that its adhesive
properties degraded fairly quickly over time. Poly 1-lysine is a
three dimensional helical polymeric molecule with side chains
containing amine functional groups which effect its adhesive
properties. With respect to one aspect of the invention, it was
postulated that the physical degradation of this three-dimensional
helical structure effectively negated the adhesive abilities of
these side chain functional groups. In an attempt to retain this
adhesive property of the poly 1-lysine coated slides, the concept
of stabilizing the poly 1-lysine molecule structure was considered
a viable solution.
[0013] In another aspect of the invention, a stabilization process
is provided to cross-link the poly 1-lysine molecule using
formaldehyde. It is believed that the formaldehyde molecules link
the functional groups on the end of the side chains back into the
molecular framework, thus physically stabilizing the
three-dimensional structure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 schematically shows a process flow for an exemplary
cell adhesion solution method as employed in accordance with the
invention.
[0015] FIG. 2 shows schematically a process for depositing a
cytological specimen on a slide processed with an exemplary cell
adhesion solution method as employed in accordance with the
invention.
[0016] FIG. 3 shows schematically a set of slides processed with an
exemplary cell adhesion solution method as employed in accordance
with the invention being stored for future use.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0017] The present invention generally relates to a cross-linked
amino acid polymer solution that can be used to create strong
cellular adhesion properties to substrates, such as glass slides,
that are stable over a long-term period. This solution allows
pre-coating of such slides such that they may be stored for an
indefinite period of time until needed.
[0018] More particularly, the solutions are aqueous and consist of
an amino acid polymer, a cross-linking agent, and a buffering agent
to maintain the pH of the solution above 6.0.
[0019] In one embodiment, slides coated with the solutions
demonstrated stronger cell adhesion properties than currently
available solutions and maintained stability up to 31 days.
[0020] Referring now to FIG. 1, there shown schematically is a
process flow 100 for an exemplary cell adhesion solution method as
employed in accordance with the invention. In one aspect of the
invention, the following steps are used:
[0021] 1. At step 10, a solution of poly 1-lysine is prepared,
[0022] 2. At step 20, neutral buffered formalin is added to the
solution of poly 1-lysine,
[0023] 3. At step 30, the resulting solution of poly 1-lysine and
neutral buffered formalin are mixed,
[0024] 4. At step 35, the mixed solution is allowed to stand for a
predetermined period of time to form a cell adhesion solution,
[0025] 4. At step 40, slides are dipped in the cell adhesion
solution, and
[0026] 5. At step 50, slides are removed from the mixture and
allowed to dry before using.
[0027] Materials Used
[0028] In implementing the process 100, the following materials
were used:
[0029] 1. Deionized H.sub.2O (DI H.sub.2O) that is prepared using a
standard laboratory Deionized H.sub.2O system.
[0030] 2. Poly L-Lysine that may be obtained, for example, from
Sigma Biochemicals company.
[0031] 3. Neutral Buffered Formalin as may be obtained, for
example, from Fisher Scientific company.
[0032] In one example, a solution of a cross-linked polymer for
substrates included an amino acid polymer, a buffered cross-linking
agent, and deionized water (DI H.sub.2O). In a preferred
embodiment, the amino acid polymer is selected from a neutral or
basic amino acid, such as but not limited to poly 1-lysine or poly
1-arginine. In another embodiment, the amino acid polymer is poly
1-1sine. In another embodiment the amino acid polymer is poly
1-1sine of molecular weight of 100,000 or higher. In another
embodiment the cross-linking agent contains an aldehyde functional
group. In another embodiment the cross-linking aldehyde agent is a
buffered formaldehyde solution. In another embodiment the
cross-linking aldehyde agent is a 10% neutral buffered formalin
solution.
[0033] In another embodiment of the invention, a solution of a
cross-linked polymer that creates strong and stable cell adhesion
properties to substrates when applied, includes by volume about
0.03% poly 1-lysine in DI H.sub.2O; and about 25% neutral buffered
formalin. In a more preferred embodiment the solution of a
cross-linked polymer includes about 0.015% poly 1-lysine in DI
H.sub.2O and about 12.5% neutral buffered formalin. In another
embodment the solution of a cross-linked polymer includes about
0.0075% poly 1-lysine in DI H.sub.2O and about 6.25% neutral
buffered formalin.
EXAMPLES
[0034] The following example formulations have proven to be
effective.
[0035] Formula #1
[0036] 1. A working stock solution of 0.03% poly 1-lysine is
prepared by dissolving 0.03 grams of poly 1-lysine in 0.100 liters
of DI H.sub.2O.
[0037] 2. Equal parts of 0.03% poly 1-lysine and neutral buffered
formalin are added to the working stock solution of 0.03% poly
1-lysine and mixed thoroughly to provide a cell adhesion
solution.
[0038] 3. The cell adhesion solution is allowed to stand for 30
minutes.
[0039] 4. The cell adhesion solution is transferred to an
appropriate container for coating of substrates, such as glass
slides.
[0040] 5. Substrates are dipped into the cell adhesion
solution.
[0041] 6. Substrates are removed from the cell adhesion solution to
dry completely (drying may take from 5 minutes to 1 hour, depending
on relative humidity),
[0042] 7. Substrates may be used immediately or stored until
needed.
[0043] Formula #2
[0044] 1. A working stock solution of 0.03% poly 1-lysine is
prepared by dissolving 0.03 grams of poly 1-lysine in 0.100 liters
of DI H.sub.2O.
[0045] 2. Two parts DI H.sub.2O is added to one part 0.03% poly
1-lysine and one part neutral buffered formalin and mixed
thoroughly to make the cell adhesion solution.
[0046] 3. The cell adhesion solution is allowed to stand for 30
minutes.
[0047] 4. The cell adhesion solution is transferred to an
appropriate container for coating of substrates, such as glass
slides.
[0048] 5. Substrates are dipped into the cell adhesion
solution.
[0049] 6. Substrates are removed from the cell adhesion solution to
dry completely (drying may take from 5 minutes to 1 hour, depending
on relative humidity),
[0050] 7. Substrates may be used immediately or stored until
needed.
[0051] In experiments conducted at TriPath Imaging, Inc. of
Burlington, N.C., slides processed with an exemplary cell adhesion
solution method as employed in accordance with the invention were
compared against conventionally processed slides. A single cervical
cytological sample was processed on all slides in the experiment.
Two slides were coated using a known formula. It was noted that
slides freshly prepared with the known formula demonstrate good
cellular adhesion, but diminished in adhesion over time. However,
slides prepared with two embodiments of the cell adhesion solution
made in accordance with the present invention, namely formula #1
and formula #2, demonstrated superior adhesion. This despite the
observation that the amino acid polymer concentration decreased two
and four fold for formula #1 and formula #2 respectively.
[0052] Referring now to FIG. 2, FIG. 2 shows schematically a
process for depositing a cytological specimen 5 on a slide 1
processed with an exemplary cell adhesion solution method as
employed in accordance with the invention. The cytological specimen
5 may be deposited using standard methods as generally indicated by
arrow 7. The cytological specimen 5 may comprise, but is not
limited to, a cervical cytological sample.
[0053] FIG. 3 shows schematically a set of slides 60 processed with
an exemplary cell adhesion solution method as employed in
accordance with the invention being stored for future use in a
storage bin 70.
[0054] The invention has been described herein in considerable
detail in order to comply with the Patent Statutes and to provide
those skilled in the art with the information needed to apply the
novel principles of the present invention, and to construct and use
such exemplary and specialized components as are required. However,
it is to be understood that the invention may be carried out by
specifically different materials and structural configurations, and
that various modifications, both as to materials and structural
configurations and operating procedures, may be accomplished
without departing from the true spirit and scope of the present
invention.
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